Function Analysis Of An UDP-glycosyltransferase Involved In The Biosynthesis Of Polyphenolic Glucoside In Camellia Sinensis

Polyphenols are one of the largest groups of compounds that confer benefits to the health of plants and humans.Flavonol glycosides are a major ingredient of polyphenols in Camellia Sinensis.A polyphenolic glycosyltransferase(CsUGT72AM1)belonging to cluster Ⅲ b was isolated from the tea plant.The enzyme assay indicated CsUGT72AM1 has characteristics of multi substrate and multi-site.The main results are as follows:1.To obtain full-length cDNA of CsUGT72AM1,3’RACE and 5’RACE clone assays were performed based on the EST sequences of CsUGT72AM1.The gene was named CsUGT72AMl.The full-length cDNA of CsUGT72AM1 is 1416 bp.It encodes 472 amino acids with a calculated molecular mass of 50.92 kDa and an isoelectric point of 5.21.By phylogenetic tree analysis,CsUGT72AM1 was divided into Cluster Ⅲ B group and existed between AtUGT72E1 and LjUGT72Z2.Therefore,that rCsUGT72AM1 can not only catalyze monolignol but also flavonol and flavanone in vitro.2.To further characterize the biological functions of CsUGT72AM1 in tea plants,the expression profiles of CsUGT72AM1 in various tissues and in different light qualities induction were analyzed by qRT-PCR.The results show that the high expression of CsUGT72AM1 in old leaves,low expression in young leaves.In addition,the expressional level of CsUGT72AM1 was significantly up-regulated under blue light,and UV light compared to white light and dark control.In addition,flavonoid and lignin compounds of various samples were detection.The content of lignin reached higher levels in old leaves and F-3-O-Glucosides(glc)reached higher levels in 3rd leaves.Moreover,lignin content and F-3-O-Glucosides(glc)compounds were increased under blue light treatment.3.PMAL-C2x-CsUGT72AM1 recombinant plasmid was constructed,and then the recombinant plasmid CsUGT72AM1 was transformed into Novablue expression strain.Finally,the fusion protein was obtained through culture and induction of the expressed strain.The fusion protein was purified to get the pure protein.HPLC and LC-MS were used to verify the activity of the recombinant protein.HPLC and LC-MS results showed that rCsUGT72AM1 exhibited the characteristics of substrate diversity.The recombinant protein not only catalyzed the formation of glucosides of flavonoids,but also catalyzed the formation of glycosides from lignin.In addition,the products of flavanone naringenin were verified by nuclear magnetic resonance technology.The results showed that rCsUGT72AM1 could catalyze naringenin to form 7-O-glycosides and 4 ’-O-glycosides in vitro.The results showed that the recombinant protein had a variety of catalytic sites.4.In order to analyze the affinity of recombinant protein rCsUGT72AM1 to different substrates,we carried out an enzyme kinetics experiment.The experimental results showed that the affinity of recombinant protein rCsUGT72AM1 to naringin was the highest,and that of Km was 28.68.In the catalytic efficiency,the recombinant protein rCsUGT72AM1 showed high catalytic efficiency for the eriodyctyol and cinnamaldehyde.